Cytochrome P450 2D6 Substrates Research Articles

Active-site structure, binding and redox activity of the heme-thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study.

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-007-0303-1) contains supplementary material, which is available to authorized users.

Pharmacokinetics of codeine and its metabolite morphine in ultra-rapid metabolizers due to CYP2D6 duplication

Codeine is an analgesic drug acting on mu-opiate receptors predominantly via its metabolite morphine, which is formed almost exclusively by the genetically polymorphic enzyme cytochrome P450 2D6 (CYP2D6). Whereas it is known that individuals lacking CYP2D6 activity (poor metabolizers, PM) suffer from poor analgesia from codeine, ultra-fast metabolizers (UM) due to the CYP2D6 gene duplication may experience exaggerated and even potentially dangerous opioidergic effects and no systematical study has been performed so far on this question. A single dose of 30 mg codeine was administered to 12 UM of CYP2D6 substrates carrying a CYP2D6 gene duplication, 11 extensive metabolizers (EM) and three PM. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism methods and a single-base primer extension method for characterization of the gene-duplication alleles. Pharmacokinetics was measured over 24 h after drug intake and codeine and its metabolites in plasma and urine were analyzed by liquid chromatography with tandem mass spectrometry. Significant differences between the EM and UM groups were detected in areas under the plasma concentration versus time curves (AUCs) of morphine with a median (range) AUC of 11 (5-17) microg h l(-1) in EMs and 16 (10-24) microg h l(-1) in UM (P=0.02). In urine collected over 12 h, the metabolic ratios of the codeine+codeine-6-glucuronide divided by the sum of morphine+its glucuronides metabolites were 11 (6-17) in EMs and 9 (6-16) in UM (P=0.05). Ten of the 11 CYP2D6 UMs felt sedation (91%) compared to six (50%) of the 12 EMs (P=0.03). CYP2D6 genotypes predicting ultrarapid metabolism resulted in about 50% higher plasma concentrations of morphine and its glucuronides compared with the EM. No severe adverse effects were seen in the UMs in our study most likely because we used for safety reasons a low dose of only 30 mg. It might be good if physicians would know about the CYP2D6 duplication genotype of their patients before administering codeine.

Catalytic Site Prediction and Virtual Screening of Cytochrome P450 2D6 Substrates by Consideration of Water and Rescoring in Automated Docking

Automated docking strategies successfully applied to binding mode predictions of ligands in Cyt P450 crystal structures in an earlier study (de Graaf et al. J. Med. Chem. 2005, 7, 2308-2318) were used for the catalytic site prediction (CSP) of 65 substrates in a CYP2D6 homology model. The consideration of water molecules at predicted positions in the active site and the rescoring of pooled docking poses from four different docking programs (AutoDock, FlexX, GOLD-Goldscore, and GOLD-Chemscore) with the SCORE scoring function enabled the successful prediction of experimentally reported sites of catalysis of more than 80% of the substrates. Three docking algorithms (FlexX, GOLD-Goldscore, and GOLD-Chemscore) were subsequently used in combination with six scoring functions (Chemscore, DOCK, FlexX, GOLD, PMF, and SCORE) to assess the ability of docking-based virtual screening methods to prioritize known CYP2D6 substrates seeded into a drug-like chemical database (in the absence and presence of active-site water molecules). Finally, the optimal docking strategy in terms of virtual screening accuracy, GOLD-Chemscore with the consideration of active-site water (60% of known substrates recovered in the top 5% of the ranked drug-like database), was verified experimentally; it was successfully used to identify high-affinity CYP2D6 ligands among a larger proprietary database.

Development of a PCR-Based Strategy for CYP2D6 Genotyping Including Gene Multiplication of Worldwide Potential Use

There is growing consensus on the potential use of pharmacogenetics in clinical practice, and hopes have been expressed for application to the improvement of global health. However, two major challenges may lead to widening the "biotechnological gap" between the developing and the industrial world; first the unaffordability of some current technologies for poorer countries, and second the necessity of analyzing all described alleles for every clinical case due to the inability to predict the ethnic group of a given patient. Because of its role in the metabolism of a number of drugs, cytochrome P450 2D6 (CYP2D6) is an excellent candidate for use in the optimization of drug therapy. CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles, and subjects can be classified as poor metabolizers (PM), extensive metabolizers (EM), or ultrarapid metabolizers (UM) of a given CYP2D6 substrate. Several strategies and methods for CYP2D6 genotyping exist. Some, however, are expensive and laborious. The aim of this study was to design a PCR-based genotyping methodology to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes for routine clinical use, independent of the individual's ethnic group. CYP2D6 is amplified in initial extra long PCRs (XL-PCRs), which subsequently undergo fragment-length polymorphism analysis for the determination of carriers of CYP2D6 allelic variants. The same XL-PCRs are also used for the determination of CYP2D6 multiplication and 2D6*5 allele (abolished activity). The application of this new strategy for the detection of CYP2D6 mutated alleles and multiplications to routine clinical analysis will enable the PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals at most locations.

The role of phenylalanine 483 in cytochrome P450 2D6 is strongly substrate dependent

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1′-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783–92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V max for bufuralol 1′-hydroxylation, while the K m was hardly affected. The V max for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K m was much larger. For dextromethorphan, in addition to dextrorphan ( O-demethylation) and 3-methoxymorphinan ( N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent.

Celecoxib is often combined with cytochrome P450 2D6 substrates in general clinical practice

Celecoxib is often combined with cytochrome P450 2D6 substrates in general clinical practice

Identification of Subtypes of CYP2D Gene Rearrangements among Carriers of CYP2D6 Gene Deletion and Duplication

Cytochrome P450 2D6 (CYP2D6) is one of the best-known polymorphic drug-metabolizing enzymes. Rapidly evolving genotyping techniques permit the identification of single-nucleotide polymorphisms (SNPs) and thereby a prediction of individual metabolic capacities for CYP2D6 substrates. A considerable part of interindividual variability in CYP2D6 enzyme activity, however, is not related to SNPs but to gene deletions and duplications. Currently used genotyping methods assume that these gene rearrangements are homogeneous. We analyzed the interindividual variability in CYP2D6 gene arrangements in genomic DNA from 740 Caucasian individuals by allele-specific PCR to identify common SNPs of the CYP2D6 gene that correspond to the variant alleles CYP2D6*3, *4, and *9. We investigated the presence and variability of CYP2D6*5 (gene deletion), CYP2D6x2 (gene duplication), and CYP2D6xn (gene amplification) by EcoRI and XbaI restriction fragment length polymorphism analyses and by long PCR plus KpnI and BamHI digestion. The presence of new mutations at the CYP2D locus was analyzed by sequencing. CYP2D6 gene rearrangements were present in >12% of individuals. Variability in the rearrangements regarding both gene deletion and gene duplication existed, and one of the unusual arrangements led to incorrect phenotype prediction. The frequency for carriers of unusual gene rearrangements was <0.3% (95% confidence interval, 0%-0.6%) in the population studied. Heterogeneity in CYP2D6 gene rearrangement exists, but the allele frequency indicates that the risk for an erroneous phenotype prediction related to such variability is extremely low and that this risk can be neglected in routine analyses.

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

CYP2D6 is of great importance for the metabolism of clinically used drugs and about 20-25% of those are metabolised by this enzyme. In addition, the enzyme utilises hydroxytryptamines as endogenous substrates. The polymorphism of the enzyme results in poor, intermediate, efficient or ultrarapid metabolisers (UMs) of CYP2D6 drugs. It is plausible that the UM genotype, where more than one active gene on one allele occurs, is the outcome of selective dietary selection in certain populations in North East Africa. The UM phenotype affects 5.5% of the population in Western Europe. A hypothesis for the evolutionary basis behind selection for CYP2D6 gene duplications is presented in relation to selection for Cyp6 variants in insecticide resistant Drosophila strains. The polymorphism of CYP2D6 significantly affects the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The consequences of the polymorphism at ordinary drug doses can be either adverse drug reactions or no drug response. Examples are presented where CYP2D6 polymorphism affects the efficacy and costs of drug treatment. Predictive CYP2D6 genotyping is estimated by the author to be beneficial for treatment of about 30-40% of CYP2D6 drug substrates, that is, for about 7-10% of all drugs clinically used, although prospective clinical studies are necessary to evaluate the exact benefit of drug selection and dosage based on the CYP2D6 genotype.

CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

1. The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized.2. Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively.3. Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (Km = 179 ± 74 μM, Clint = 0.41 ± 0.26 ml mg−1 h−1) and the PM subject (Km = 225 μM, Clint = 0.19 ml mg−1 h−1) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects.4. CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug–drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.

Validation of model of cytochrome P450 2D6: an in silico tool for predicting metabolism and inhibition.

There has been much interest in the development of a predictive model of cytochrome P450 2D6 particularly because this enzyme is involved in the oxidation of at least 50 drugs. Previously we have described the combined use of homology modeling and molecular docking to correctly position a range of substrates in the CYP2D6 active site with the known sites of metabolism above the heme. Here, our approach identifies correctly the site of metabolism of the atypical (no basic nitrogen) cytochrome P450 2D6 substrate, spirosulfonamide. The same method is used to screen a small compound database for cytochrome P450 2D6 inhibition. A database containing 33 compounds from the National Cancer Institute database was docked into our cytochrome P450 2D6 homology model using the program GOLDv2.0. Experimental IC50 values for the 33 compounds were determined; comparison with the corresponding docked scores revealed a correlation with a regression coefficient of r2 = 0.61 (q2 = 0.59). The method was able to discriminate between tight and weak binding compounds and correctly identified several novel inhibitors. The results therefore suggest that our approach, which combines homology modeling with molecular docking, has produced a useful predictive in silico tool for cytochrome P450 2D6 inhibition, which is best used as one filter in a multifilter database screen.

Impact of CYP2D6 intermediate metabolizer alleles on single-dose desipramine pharmacokinetics.

This study utilized cytochrome P450 2D6 (CYP2D6) genotypes to explain variability of desipramine pharmacokinetics in a cohort of non-poor metabolizer individuals. In an interaction study utilizing desipramine as a probe, genotyping for the CYP2D6*3, *4, *5 and *6 alleles was used to screen out CYP2D6 poor metabolizers. Individuals were categorized according to these and additional alleles (CYP2D6*2, *9, *10, *17, *41 and x2). Genotypes of individuals heterozygous for two or three of *2, *17 and *41 alleles were confirmed by molecular haplotyping. Pharmacokinetic parameters of desipramine were analysed according to CYP2D6 category. Molecular haplotyping was necessary to definitively categorize four of 16 individuals. A subject who had unusually high plasma elimination half-time, exposure and metabolic ratios carried an intermediate metabolizer (IM) *9 allele in combination with a non-functional allele. This combination has a population frequency of less than 1 : 200. Individuals with *1/*1, *1/*2 and *2/*2 genotypes had lower than average plasma elimination half-time, exposure and metabolic ratios. For desipramine, additional genotyping of CYP2D6 IM alleles helped define subgroups of the CYP2D6-positive cohort. This suggests that genotyping for IM alleles will aid in interpretation of clinical trials involving CYP2D6 substrates. Due to the diversity of IM alleles, molecular haplotyping may be necessary to fully characterize CYP2D6 genotype-phenotype relationships.

Human liver cytochrome P450 2D6 genotype, full-length messenger ribonucleic acid, and activity assessed with a novel cytochrome P450 2D6 substrate*1

Human liver cytochrome P450 2D6 genotype, full-length messenger ribonucleic acid, and activity assessed with a novel cytochrome P450 2D6 substrate*1

Polymorphic Cytochrome P450 2D6: Humanized Mouse Model and Endogenous Substrates

Cytochrome P450 2D6 (CYP2D6) is the first well‐characterized polymorphic phase I drug‐metabolizing enzyme, and more than 80 allelic variants have been identified for the CYP2D6 gene, located on human chromosome 22q13.1. Human debrisoquine and sparteine metabolism is subdivided into two principal phenotypes—extensive metabolizer and poor metabolizer—that arise from variant CYP2D6 genotypes. It has been estimated that CYP2D6 is involved in the metabolism and disposition of more than 20% of prescribed drugs, and most of them act in the central nervous system or on the heart. These drug substrates are characterized as organic bases containing one nitrogen atom with a distance about 5, 7, or 10 Å from the oxidation site. Aspartic acid 301 and glutamic acid 216 were determined as the key acidic residues for substrate‐enzyme binding through electrostatic interactions. CYP2D6 transgenic mice, generated using a lambda phage clone containing the complete wild‐type CYP2D6 gene, exhibits enhanced metabolism and disposition of debrisoquine. This transgenic mouse line and its wild‐type control are models for human extensive metabolizers and poor metabolizers, respectively, and would have broad application in the study of CYP2D6 polymorphism in drug discovery and development, and in clinical practice toward individualized drug therapy. Endogenous 5‐methoxyindolethylamines derived from 5‐hydroxytryptamine were identified as high‐affinity substrates of CYP2D6 that catalyzes their O‐demethylations with high enzymatic capacity and specificity. Thus, polymorphic CYP2D6 may play an important role in the interconversions of these psychoactive tryptamines, including a crucial step in a serotonin‐melatonin cycle.

Comparative molecular field analysis and QSAR on substrates binding to cytochrome P450 2D6

In this study, we utilized comparative molecular field analysis (CoMFA) to gain a better understanding of the steric and electrostatic features of the cytochrome P450 2D6 (CYP2D6) active site. The training set consists of 24 substrates with reported K M values from liver microsomal CYP2D6 spanning an activity range of almost three log units. The low energy conformers were fit by root mean square (RMS) to minaprine at the site of metabolism and to the protonated nitrogen. In this manner, we constructed two CoMFA models, one model with a distance constraint and another without. The model with the distance parameter (non-crossvalidated R 2=0.99) was approximately equal to the CoMFA without a distance parameter (non-cross-validated R 2=0.98). Validation of our CoMFA was accomplished by predicting the K M values of 15 diverse CYP2D6 substrates not in the original training set resulting in a predictive R 2=0.62. Finally, we also pursued correlations of p K a and log P with CYP2D6 substrate K M in an effort to investigate other physicochemical properties.

Dietary levels of quinine in tonic water do not inhibit CYP2D6 in vivo

Quinine is a bitter alkaloid that is used as a flavoring agent in tonic water. Studies suggest that quinine can inhibit cytochrome P450 2D6 (CYP2D6) which could have implications for the metabolism of co-ingested drugs. We conducted a study with 11 healthy volunteers (7 men, 4 women; aged 26–54). After urinary void, each subject consumed either 1000 ml of carbonated water or 1000 ml of tonic water containing 80 mg quinine in a crossover design. Following each beverage subjects ingested an oral dose of 30 mg dextromethorphan (DM). Urine was collected for 8 h and analyzed for DM and dextrophran, its CYP2D6 mediated metabolite. The ratio of DM and its metabolite is an established measure of CYP2D6 activity. All subjects metabolized the vast majority of DM to its metabolite after both the carbonated water and the tonic water. The ratio (mean±S.D.) of DM to its metabolite was 0.013±0.028 after the carbonated water and 0.032±0.067 after the quinine containing water. No significant difference in the ratios was observed between the two beverages ( P>0.05). We conclude that quinine as consumed in tonic water does not inhibit CYP2D6 activity in vivo. Thus, quinine should not alter the metabolism of CYP2D6 substrates taken concomitantly with tonic water.

Duloxetine is both an inhibitor and a substrate of cytochrome P4502D6 in healthy volunteers.

Duloxetine, a potent dual reuptake inhibitor of serotonin and norepinephrine currently undergoing clinical investigation for treatment of depression and stress urinary incontinence, has the potential to act as both a substrate and an inhibitor of cytochrome P4502D6 (CYP2D6). Our objectives were to determine the effect of duloxetine on the pharmacokinetics of desipramine, a tricyclic antidepressant metabolized by CYP2D6 (study 1), and the effect of paroxetine, a potent CYP2D6 inhibitor, on duloxetine pharmacokinetics (study 2). Subjects were healthy men and women between 21 and 63 years old. All subjects were genotypically CYP2D6 extensive metabolizers. In study 1, 50 mg of desipramine was administered as a single dose alone and in the presence of steady-state duloxetine 60 mg twice daily. In study 2, steady-state pharmacokinetics of duloxetine 40 mg once daily were determined in the presence and absence of steady-state paroxetine 20 mg once daily. Duloxetine increased the maximum plasma concentration of desipramine 1.7-fold and the area under the concentration-time curve 2.9-fold. Paroxetine increased the maximum plasma concentration of duloxetine and the area under the concentration-time curve at steady state 1.6-fold. Reports of adverse events were similar whether duloxetine was administered alone or in combination with desipramine or paroxetine. Duloxetine 60 mg twice daily is a moderately potent CYP2D6 inhibitor, intermediate between paroxetine and sertraline. The potent CYP2D6 inhibitor paroxetine has a moderate effect on duloxetine concentrations. The results of these 2 studies suggest that caution should be used when CYP2D6 substrates and inhibitors are coadministered with duloxetine.

Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis.

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Patient-Tailored Antiemetic Treatment With 5-Hydroxytryptamine Type 3 Receptor Antagonists According to Cytochrome P-450 2D6 Genotypes

The use of serotonin 5-hydroxytryptamine type 3 receptor antagonists has substantially reduced, but not eliminated, nausea and vomiting in cancer chemotherapy. This study sought to investigate whether efficacy of antiemetic treatment with ondansetron and tropisetron depends on cytochrome P-450 2D6 (CYP2D6) genotype, hypothesizing that the rapid and particularly the ultrarapid metabolizers of these drugs are at risk of being undertreated. Included in the study were 270 cancer patients receiving their first day of chemotherapy. Nausea and vomiting were documented using standardized interviews. The intensity of nausea was measured with visual analog scales before and twice during the chemotherapy. The relationship between the CYP2D6 genotypes and the tropisetron serum concentrations 3 and 6 hours after drug administration was analyzed in a subgroup of 42 patients. CYP2D6 genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism analysis. Genetically defined poor metabolizers had higher serum concentrations of tropisetron than all other patients (P <.03). Approximately 30% of all patients receiving chemotherapy experienced nausea and vomiting. Genetically defined ultrarapid meta-bolizers of CYP2D6 substrates had higher frequency of vomiting within the first 4 hours (P <.001) and within the period 5 to 24 hours (P <.03) after treatment than all the other patients; the tendency for nausea was similar. This difference was more pronounced in patients treated with tropisetron than in those treated with ondansetron. Antiemetic treatment with tropisetron or ondansetron could be improved by adjustment for the CYP2D6 genotype; approximately 50 subjects would have to be genotyped to protect one patient from severe emesis.

Coumarin Substrates for Cytochrome P450 2D6 Fluorescence Assays

A set of nine 4-aminomethyl-7-alkoxycoumarin derivatives was synthesized and characterized as substrates for O-dealkylation by recombinant cytochrome P450 2D6, a major human enzyme involved in drug metabolism. Enzymatic O-dealkylation yields 7-hydroxycoumarins, which have useful fluorescence properties. The substrates, which differed in substitution at the amino and 7-hydroxy positions, varied in terms of catalytic efficiency of O-dealkylation and in their selectivity as substrates for cytochrome P450 2D6 in human liver microsomes. Several of the compounds are useful as cytochrome P450 2D6 substrates in single-phase, rapid-throughput assays.